DOCSLIB.ORG

Short- and Long-Term Impact of Hyperoxia on the Blood and Retinal Cells’ Transcriptome in a Mouse Model of Oxygen- Induced Retinopathy

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Short- and Long-Term Impact of Hyperoxia on the Blood and Retinal Cells’ Transcriptome in a Mouse Model of Oxygen- Induced Retinopathy www.nature.com/pr BASIC SCIENCE ARTICLE OPEN Short- and long-term impact of hyperoxia on the blood and retinal cells’ transcriptome in a mouse model of oxygen- induced retinopathy Magdalena Zasada1, Anna Madetko-Talowska2, Cecilie Revhaug3,4, Anne Gro W. Rognlien3,4, Lars O. Baumbusch3,Teofila Książek2, Katarzyna Szewczyk2, Agnieszka Grabowska2, Miroslaw Bik-Multanowski2, Jacek Józef Pietrzyk1, Przemko Kwinta1 and Ola Didrik Saugstad3,4 BACKGROUND: We aimed to identify global blood and retinal gene expression patterns in murine oxygen-induced retinopathy (OIR), a common model of retinopathy of prematurity, which may allow better understanding of the pathogenesis of this severe ocular prematurity complication and identification of potential blood biomarkers. METHODS: A total of 120 C57BL/6J mice were randomly divided into an OIR group, in which 7-day-old pups were maintained in 75% oxygen for 5 days, or a control group. RNA was extracted from the whole-blood mononuclear cells and retinal cells on days 12, 17, and 28. Gene expression in the RNA samples was evaluated with mouse gene expression microarrays. RESULTS: There were 38, 1370 and 111 genes, the expression of which differed between the OIR and control retinas on days 12, 17, and 28, respectively. Gene expression in the blood mononuclear cells was significantly altered only on day 17. Deptor and Nol4 genes showed reduced expression both in the blood and retinal cells on day 17. 1234567890();,: CONCLUSION: There are sustained marked changes in the global pattern of gene expression in the OIR mice retinas. An altered expression of Deptor and Nol4 genes in the blood mononuclear cells requires further investigation as they may indicate retinal neovascularization. Pediatric Research (2020) 87:485–493; https://doi.org/10.1038/s41390-019-0598-y INTRODUCTION oxygen-induced retinopathy (OIR).5 In mice, the retinas are mostly Retinopathy of prematurity (ROP), a proliferative retinal vascular avascular after birth, and retinal vascularization occurs in the early disorder affecting premature infants, is currently a major cause of neonatal period, resembling situation observed in the prematurely potentially preventable blindness in children.1 It is estimated that born infants. 5-day long continuous exposure to high oxygen ROP affects 33.2% of neonates with birth weight < 1500 g.2 ROP levels imitates the biphasic retinal vascular response observed in develops in two phases; phase 1 is initiated by hyperoxia and the young infants with ROP.6 Up till now, exploration of the retinal exacerbated by lack of the essential factors provided in utero, gene expression pattern in the murine OIR model was assessed followed by a destruction of existing vessels and arrest of normal partially. Sato et al.7 evaluated only a selected set of genes related vessel development. Phase 2 is driven by increased metabolic to angiogenesis and inflammation. Subsequent studies investi- activity of the hypoxic retina, which leads to both revascularization gated expression of the entire genome, but at the selected time and pathological neovascularization.3 An excessive abnormal points—Ishikawa et al.8 and Yang et al.9 determined a profile of compensatory vessel growth may result in fibrous scar develop- gene expression in murine retinas soon after end of hyperoxia, ment, and ultimately leads to retinal detachment.4 We believe that whereas Recchia et al.10 additionally verified gene expression in better understanding of the pathomechanisms behind ROP rodent retinas during neovascularization phase. The goal of this coupled with an early and effective method of the risk study was to identify potential ROP blood biomarkers and provide stratification of ROP development would allow to take true a more molecular-based understanding of ROP by comparing the preventive actions and/or targeted treatment. Genome-wide gene expression profile at three time points (vaso-obliteration microarray analysis provides a simultaneous examination of the (P12), maximal neovascularization (P17) and during normalization expression of thousands of genes in a single experiment to create of the retinal damage (P28)) in the blood mononuclear cells and a comprehensive picture of dysregulated genes and pathways. A retinas obtained from the mice with and without OIR. We possibility to use the animal model that mimics key features of the hypothesized that supplemental oxygen as compared with air disease provides invaluable tool for studying ROP underlying induced gene expression changes in the blood and retinal cells, mechanisms not only in the blood, but also in the target tissue which varied depending on the length of time following exposure such as retina. The most widely used model is a murine model of to hyperoxia. 1Department of Paediatrics, Jagiellonian University Medical College, Krakow, Poland; 2Department of Medical Genetics, Jagiellonian University Medical College, Krakow, Poland; 3Department of Paediatric Research, Oslo University Hospital Rikshospitalet, Oslo, Norway and 4University of Oslo, Oslo, Norway Correspondence: Przemko Kwinta ([email protected]) These authors contributed equally: M. Zasada, A. Madetko-Talowska Received: 3 June 2019 Revised: 4 September 2019 Accepted: 6 September 2019 Published online: 2 October 2019 © The Author(s) 2019 Short- and long-term impact of hyperoxia on the blood and retinal cells’... M Zasada et al. 486 METHODS suspended by manual pipetting, then the samples were placed Murine model of OIR immediately at −80 °C. Total RNA was isolated with the use of The Norwegian Animal Research Authority approved all experi- Mouse RiboPure™-Blood RNA Isolation Kit (Invitrogen; Thermo ment protocols. The animals were cared for and handled in Fisher Scientific) according to the manufacturer’s instructions. RNA accordance with the Norwegian Legislation and Directive. OIR was concentration was measured with the use of NanoDrop Spectro- induced in C57BL/6 mice (received from: Jackson Laboratories, Bar photometer (NanoDrop ND-1000; Thermoscientific, Waltham), and Harbor, ME) according to the protocol described by Smith et al.6 RNA quality was determined by TapeStation 4200 (Agilent, Santa Briefly, mice were housed and bred at 24 °C at a 12:12 light/dark Clara). cycle with access to a standard rodent pellets and water ad libitum. On the postnatal day 7 (P7), all pups within each litter Obtaining retinas and total RNA isolation were earmarked with use of surgical micro-scissors. When a pup Whole retinas free of additional tissues were isolated immedi- was picked up from the cage, it received a number drawn by an ately under a dissecting microscope, and put into vials with assistant from blinded tickets containing numbers from 1 to 8. The RNAlater® Stabilization Solution (Thermo Fisher Scientific), kept procedure was repeated until every pup had its own number for 24 h in + 4°Candstoredina−80 °C freezer until further use. within the litter. The smallest pups (according to their body weight To isolate sufficient total RNA for microarray analysis, two retinas on P7) out of litters of >8, or any abnormal pups (with visible fromthesamemousewerepooled.Retinatissueswerethen morphologic abnormalities) were excluded from the experiment. disrupted with the use of TissueRuptor handheld rotor-stator Each litter with a nursing mother was then randomized either to homogenizer (Qiagen, Hilden, Germany) and total RNA from the hyperoxia (75% oxygen) or normoxia (21% oxygen) conditions retina cells was processed by RNeasy Micro Kit (Qiagen, Hilden, (also by drawing a ticket either with a symbol N for normoxia or H Germany) as described by the manufacturer. RNA concentration for hyperoxia), then transferred in their cage into A-Chambers was measured with the use of NanoDrop Spectrophotometer (BioSpherix Ltd, Parish, NY), where they were kept until day 12 (NanoDrop ND-1000; Thermoscientific, Waltham), and RNA (P12). Seventy-five percent oxygen concentration was achieved quality was determined by TapeStation 4200 (Agilent, Santa with a conventional oxygen mixer (oxygen (AGA AS, Oslo, Norway) Clara). plus room air). Each chamber was continuously monitored for oxygen concentration (O2-monitor, ProOX110 BioSpherix Ltd, Validation of the model Parish, NY), temperature and humidity (Thermometer/Hygrometer Eyes from randomly selected 36 pups both from normoxia and Clas Ohlson, Insjon, Sweden), and CO2 level (ProCO2 P120, hyperoxia groups, 1/3 from each harvesting time point, were used BioSpherix Ltd, Parish, NY). An open water source kept air for the verification of the model, according to the methods humidity at 40–50%. Every day there was a 10–20 min break in described in details elsewhere.3 Briefly, the eye was enucleated oxygen supply for airing the chamber and checking the mice. and fixed with 4% Paraformaldehyde Solution (Boster Biological Nursing dams stayed with their own litters throughout the entire Technology, Pleasanton CA). Then under the dissecting micro- experiment. The cages containing pups with their mothers were scope the retina was isolated, incubated in 0.5% Triton X-100 removed from the hyperoxia/normoxia chambers on day P12. (Sigma, St. Louis, MO) and stained in the lectin solution with Alexa Before removing cages with mice from A-Chambers, one more Fluor 594–I21413, Molecular Probes. Then under a dissecting randomization was performed; namely tickets with symbols, microscope retinas were mounted on a microscope slide and indicating the predicted harvesting day and type of analyses cover slipped with SlowFade medium (Thermo Fisher
Load more

Recommended publications
  • Multi-Ancestry Genome-Wide Gene-Sleep Interactions Identify Novel
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.29.123505; this version posted May 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Multi-ancestry genome-wide gene-sleep interactions identify novel loci for blood pressure Heming Wang1,2,*, Raymond Noordam3,* , Brian E Cade1,2,*, Karen Schwander4,*, Thomas W Winkler5,*, Jiwon Lee1,*, Yun Ju Sung4,*, Amy R. Bentley6,*, Alisa K Manning2,7, Hugues Aschard8,9, Tuomas O Kilpeläinen10,11, Marjan Ilkov12, Michael R Brown13, Andrea R Horimoto14, Melissa Richard15, Traci M Bartz16, Dina Vojinovic17,18, Elise Lim19, Jovia L Nierenberg20, Yongmei Liu21, Kumaraswamynaidu Chitrala22, Tuomo Rankinen23, Solomon K Musani24, Nora Franceschini25, Rainer Rauramaa26, Maris Alver27,28, Phyllis Zee29, Sarah E Harris30, Peter J van der Most31, Ilja M Nolte31, Patricia B Munroe32,33, Nicholette D Palmer34, Brigitte Kühnel35,36, Stefan Weiss37,38, Wanqing Wen39, Kelly A Hall40, Leo-Pekka Lyytikäinen41,42, Jeff O'Connell43,44, Gudny Eiriksdottir12, Lenore J Launer22, Paul S de Vries13, Dan E Arking45, Han Chen13,46, Eric Boerwinkle13,47, Jose E Krieger14, Pamela J Schreiner48, Stephen S Sidney49, James M Shikany50, Kenneth Rice51, Yii-Der Ida Chen52, Sina A Gharib53, Joshua C Bis54, Annemarie I Luik17, M Arfan Ikram17,55, André G Uitterlinden17, Najaf Amin17, Hanfei Xu19, Daniel Levy19,56, Jiang He20, Kurt
    [Show full text]
  • Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-Like Mouse Models: Tracking the Role of the Hairless Gene
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene Yutao Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Liu, Yutao, "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino- like Mouse Models: Tracking the Role of the Hairless Gene. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1824 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yutao Liu entitled "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Brynn H. Voy, Major Professor We have read this dissertation and recommend its acceptance: Naima Moustaid-Moussa, Yisong Wang, Rogert Hettich Accepted for the Council: Carolyn R.
    [Show full text]
  • RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases
    G C A T T A C G G C A T genes Review RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases Amber Willbanks, Shaun Wood and Jason X. Cheng * Department of Pathology, Hematopathology Section, University of Chicago, Chicago, IL 60637, USA; [email protected] (A.W.); [email protected] (S.W.) * Correspondence: [email protected] Abstract: Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases. Keywords: 5’ cap (5’ cap); 7-methylguanosine (m7G); R-loops; N6-methyladenosine (m6A); RNA editing; A-to-I; C-to-U; 2’-O-methylation (Nm); 5-methylcytosine (m5C); NOL1/NOP2/sun domain Citation: Willbanks, A.; Wood, S.; (NSUN); MYC Cheng, J.X. RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases. Genes 2021, 12, 627.
    [Show full text]
  • Function of Deptor and Its Roles in Hematological Malignancies
    www.aging-us.com AGING 2021, Vol. 13, No. 1 Review Function of Deptor and its roles in hematological malignancies Mario Morales-Martinez1, Alan Lichtenstein2, Mario I. Vega1,2 1Molecular Signal Pathway in Cancer Laboratory, UIMEO, Oncology Hospital, Siglo XXI National Medical Center, IMSS, México, México 2Department of Medicine, Hematology-Oncology Division, Greater Los Angeles VA Healthcare Center, UCLA Medical Center, Jonsson Comprehensive Cancer Center, Los Angeles, CA 90024, USA Correspondence to: Mario I. Vega; email: [email protected] Keywords: Deptor, Multiple Myeloma, leukemia, Non-Hodgkin Lymphoma, hematological malignances Received: November 6, 2020 Accepted: December 10, 2020 Published: January 7, 2020 Copyright: © 2020 Morales-Martinez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Deptor is a protein that interacts with mTOR and that belongs to the mTORC1 and mTORC2 complexes. Deptor is capable of inhibiting the kinase activity of mTOR. It is well known that the mTOR pathway is involved in various signaling pathways that are involved with various biological processes such as cell growth, apoptosis, autophagy, and the ER stress response. Therefore, Deptor, being a natural inhibitor of mTOR, has become very important in its study. Because of this, it is important to research its role regarding the development and progression of human malignancies, especially in hematologic malignancies. Due to its variation in expression in cancer, it has been suggested that Deptor can act as an oncogene or tumor suppressor depending on the cellular or tissue context.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN
    [CANCER RESEARCH 61, 3741–3749, May 1, 2001] Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN Mieko Matsushima-Nishiu, Motoko Unoki, Kenji Ono, Tatsuhiko Tsunoda, Takeo Minaguchi, Hiroyuki Kuramoto, Masato Nishida, Toyomi Satoh, Toshihiro Tanaka, and Yusuke Nakamura1 Laboratories of Molecular Medicine [M. M-N., M. U., K. O., T. M., T. Ta., Y. N.] and Genome Database [T. Ts.], Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Department of Obstetrics and Gynecology, School of Medicine, Kitasato University, Sagamihara 228-8555, Japan [H. K.]; Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8576, Japan [M. N.]; and Department of Obstetrics and Gynecology, Ibaraki Seinan Central Hospital, Tsukuba 306-0433, Japan [T. S.] ABSTRACT Akt/protein kinase B, cell survival, and cell proliferation (8). Over- expression of PTEN can decrease cell proliferation and tumorigenicity The PTEN tumor suppressor gene encodes a multifunctional phospha- (9, 10), an observation attributed to the ability of PTEN to induce cell tase that plays an important role in inhibiting the phosphatidylinositol-3- cycle arrest and apoptosis (11, 12). kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced ex- Thus, lack of PTEN expression may affect a complex set of pression of PTEN in various cancer cell lines decreases cell proliferation transcriptional targets. However, no systematic assessment of PTEN- through arrest of the cell cycle, accompanied in some cases by induction regulated targets in cancer cells has been reported to date.
    [Show full text]
  • Structural and Functional Analysis of Muskelin and Other Kelch-Repeat Proteins
    structural and Functional Analysis of Muskelin by Soren Prag Hansen A Thesis submitted to the University College London for the degree of PhD September 2003 Department of Biochemistry and Molecular Biology University College London Gower Street London UK ProQuest Number: U643396 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643396 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Muskelin is an intracellular, kelch-repeat protein that is functionally involved in cell spreading on thrombospondin-1. The aim of this thesis project was to investigate the role of muskelin domains in the subcellular distribution and the regulation of the protein using cellular, biochemical, and bioinformatic approaches. Using bioinformatics to compare muskelin orthologues from Mus musculus, Homo sapiens, Rattus norvegicus, Danio rerio, Drosophila meianogaster, and Anopheles gambiae, highly conserved regions within muskelin were identified, with the combination of a discoidin domain, a LisH motif, a C-terminal to LisH motif, and six kelch repeats. A further bioinformatic analysis of the kelch repeat proteins in whole genomes from Homo sapiens, Drosophila meianogaster, and Anopheles gambiae, demonstrated that muskelin has a unique molecular architecture amongst the large family of kelch repeat proteins.
    [Show full text]
  • TAF10 Complex Provides Evidence for Nuclear Holo&Ndash;TFIID Assembly from Preform
    ARTICLE Received 13 Aug 2014 | Accepted 2 Dec 2014 | Published 14 Jan 2015 DOI: 10.1038/ncomms7011 OPEN Cytoplasmic TAF2–TAF8–TAF10 complex provides evidence for nuclear holo–TFIID assembly from preformed submodules Simon Trowitzsch1,2, Cristina Viola1,2, Elisabeth Scheer3, Sascha Conic3, Virginie Chavant4, Marjorie Fournier3, Gabor Papai5, Ima-Obong Ebong6, Christiane Schaffitzel1,2, Juan Zou7, Matthias Haffke1,2, Juri Rappsilber7,8, Carol V. Robinson6, Patrick Schultz5, Laszlo Tora3 & Imre Berger1,2,9 General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID—a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)— assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8–TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core–TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo–TFIID, regulated by nuclear import of preformed cytoplasmic submodules. 1 European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France. 2 Unit for Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France. 3 Cellular Signaling and Nuclear Dynamics Program, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404 Illkirch, France.
    [Show full text]
  • Structure and Mechanism of the RNA Polymerase II Transcription Machinery
    Downloaded from genesdev.cshlp.org on October 9, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Structure and mechanism of the RNA polymerase II transcription machinery Allison C. Schier and Dylan J. Taatjes Department of Biochemistry, University of Colorado, Boulder, Colorado 80303, USA RNA polymerase II (Pol II) transcribes all protein-coding ingly high resolution, which has rapidly advanced under- genes and many noncoding RNAs in eukaryotic genomes. standing of the molecular basis of Pol II transcription. Although Pol II is a complex, 12-subunit enzyme, it lacks Structural biology continues to transform our under- the ability to initiate transcription and cannot consistent- standing of complex biological processes because it allows ly transcribe through long DNA sequences. To execute visualization of proteins and protein complexes at or near these essential functions, an array of proteins and protein atomic-level resolution. Combined with mutagenesis and complexes interact with Pol II to regulate its activity. In functional assays, structural data can at once establish this review, we detail the structure and mechanism of how enzymes function, justify genetic links to human dis- over a dozen factors that govern Pol II initiation (e.g., ease, and drive drug discovery. In the past few decades, TFIID, TFIIH, and Mediator), pausing, and elongation workhorse techniques such as NMR and X-ray crystallog- (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis raphy have been complemented by cryoEM, cross-linking for Pol II transcription regulation has advanced rapidly mass spectrometry (CXMS), and other methods. Recent in the past decade, largely due to technological innova- improvements in data collection and imaging technolo- tions in cryoelectron microscopy.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • Dynamic Modelling of the Mtor Signalling Network Reveals Complex
    www.nature.com/scientificreports OPEN Dynamic modelling of the mTOR signalling network reveals complex emergent behaviours conferred by Received: 24 August 2017 Accepted: 1 December 2017 DEPTOR Published: xx xx xxxx Thawfeek M. Varusai1,4 & Lan K. Nguyen2,3,4 The mechanistic Target of Rapamycin (mTOR) signalling network is an evolutionarily conserved network that controls key cellular processes, including cell growth and metabolism. Consisting of the major kinase complexes mTOR Complex 1 and 2 (mTORC1/2), the mTOR network harbours complex interactions and feedback loops. The DEP domain-containing mTOR-interacting protein (DEPTOR) was recently identifed as an endogenous inhibitor of both mTORC1 and 2 through direct interactions, and is in turn degraded by mTORC1/2, adding an extra layer of complexity to the mTOR network. Yet, the dynamic properties of the DEPTOR-mTOR network and the roles of DEPTOR in coordinating mTORC1/2 activation dynamics have not been characterised. Using computational modelling, systems analysis and dynamic simulations we show that DEPTOR confers remarkably rich and complex dynamic behaviours to mTOR signalling, including abrupt, bistable switches, oscillations and co-existing bistable/oscillatory responses. Transitions between these distinct modes of behaviour are enabled by modulating DEPTOR expression alone. We characterise the governing conditions for the observed dynamics by elucidating the network in its vast multi-dimensional parameter space, and develop strategies to identify core network design motifs underlying these dynamics. Our fndings provide new systems-level insights into the complexity of mTOR signalling contributed by DEPTOR. Discovered in the early 1990s as an anti-fungal agent produced by the soil bacterium Streptomyces hygroscopicus, rapamycin has continually surprised scientists with its diverse clinical efects including potent immunosuppres- sive and anti-tumorigenic properties1–3.
    [Show full text]